Evaluation of the total oxy-radical scavenging capacity of catechins isolated from green tea

Food Chemistry 121 (2010) 1089–1094

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Evaluation of the total oxy-radical scavenging capacity of catechins isolated from green tea Keon Wook Kang a, Soo Jin Oh b, Shi Yong Ryu c, Gyu Yong Song b, Bong-Hee Kim b, Jong Seong Kang b, Sang Kyum Kim b,* a b c

College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea College of Pharmacy and RCTCP, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejeon 305-764, Republic of Korea Korea Research Institute of Chemical Technology, Daejeon 305-606, Republic of Korea

a r t i c l e

i n f o

Article history: Received 21 October 2009 Received in revised form 24 November 2009 Accepted 26 January 2010

Keywords: Catechins Antioxidant Green tea Oxidative stress Oxy-radicals

a b s t r a c t The antioxidant activity of catechins isolated from green tea against peroxyl radicals, hydroxyl radicals, and peroxynitrite was determined using the total oxy-radical scavenging capacity (TOSC) assay. ()-Epicatechin, ()-epigallocatechin, ()-epicatechin-3-gallate, and ()-epigallocatechin-3-gallate were isolated and their structures were characterised based on their physical and spectral properties, and by comparison of these results with similar data in the literature. ()-Epigallocatechin showed the highest TOSC value, and ()-epicatechin-3-gallate was the least effective among the catechins tested. These results indicated that the presence of a gallate group at the three position plays a critical role in their antioxidant activity. However, additional insertion of a hydroxyl group at the 50 position in the B ring attenuates the oxy-radical scavenging capacity of catechins. These results suggest that the antioxidant activity of catechins is dependent on the reactivity of both the original catechins and their products generated during reaction with oxy-radicals. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Green tea is a widely used beverage and a rich source of catechin polyphenols. Numerous experimental and epidemiological studies support the health benefits of green tea consumption, including chemopreventive properties (Na & Surh, 2008), antiinflammatory effects (Tedeschi, Suzuki, & Menegazzi, 2002), neuroprotective effects (Amit, Avramovich-Tirosh, Youdim, & Mandel, 2008), and antihypertensive effects (Babu & Liu, 2008). The major polyphenols present in green tea are ()-epicatechin (EC), ()-epigallocatechin (EGC), ()-epicatechin-3-gallate (ECG), and ()-epigallocatechin-3-gallate (EGCG), which comprise 30–42% of solid green tea extract (Mukhtar & Ahmad, 1999). EGCG, the most abundant polyphenol in green tea, inhibits activation of nuclear factorjB and AP-1 in response to inflammatory stimuli (Na & Surh, 2008). EGCG can enhance the cellular defence activities by inducing antioxidant and detoxifying enzymes via activation of nuclear factor erythroid 2 p45-related factor (Nrf2) (Na & Surh, 2008). These results suggest that catechins provide beneficial health effects via regulation of cellular signalling and gene expression. Catechins effectively scavenge oxy-radicals involved in the pathogenesis of many chronic diseases, including cardiovascular * Corresponding author. Tel.: +82 42 821 5930; fax: +82 42 823 6566. E-mail address: [email protected] (S.K. Kim). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.01.055

diseases, cancer, neurodegenerative diseases, and ageing. Drinking green or black tea is associated with an increased plasma total antioxidant capacity determined using the ferric reducing ability of plasma assay in human subjects (Leenen, Roodenburg, Tijburg, & Wiseman, 2000). In addition, drinking 900 ml of green tea daily for 7 days reduced 8-hydroxy-20 -deoxyguanosine, a biomarker of oxidative stress on DNA, and malondialdehyde, a biomarker of lipid peroxidation, in smokers (Klaunig et al., 1999). These results indicate the ability of green tea to improve the overall antioxidative status and to protect against oxidative stress. To investigate the structure–activity relationship between catechins and their antioxidant properties, we evaluated the antioxidant capacities of (+)-catechin (C), EC, EGC, ECG, EGCG, and gallic acid (GA) against peroxyl radicals, hydroxyl radicals, and peroxynitrite using the total oxy-radical scavenging capacity (TOSC) assay. The TOSC assay, developed by Regoli and Winston (1999), is a method for evaluating antioxidant activity based on the inhibition of oxy-radical-induced ethylene gas production from a-keto-cmethiolbutyric acid (KMBA). The advantages of this method are that it is simple, rapid, and applicable for both biological tissues and pure antioxidants (Kim et al., 2009; Tung, Ding, Kim, Bae, & Kim, 2008). Furthermore, unlike the 2,2-diphenyl-2-picrylhydrazyl (DPPH) assay, this method can evaluate antioxidant capacity against physiological oxidants, including peroxynitrite and hydroxyl radicals. Therefore, it is of interest to determine the oxy-radical

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scavenging capacity of the catechins in this assay system and to assign quantifiable values to their antioxidant activities. 2. Materials and methods 2.1. Materials Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), GA, C, KMBA, diethylenetriamine-pentaacetic acid (DTPA), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), ascorbic acid, ferrous ammonium sulphate, and 2,20 -azobis-amidinopropane (ABAP) were purchased from Sigma–Aldrich (St. Louis, MO). EC, EGC, ECG, and EGCG were isolated from green tea and the purity of the isolated catechins was higher than 99%. All other chemicals and solvents were of reagent grade or better. 2.2. Extraction and isolation The leaves of green tea (20 kg) were collected at Boseong-gun, Jeonnam-do, Korea. The leaves of green tea were dried and extracted with methanol (50 l) by standing at room temperature for 1 week. The methanol extract (7 kg) was suspended in water (5 l) and washed out twice with n-hexane. The remaining water layer was extracted successively with ethylacetate and butanol (each 2 l  3) to give an ethylacetate extract (622 g) and a butanol extract (1942 g). The butanol extract (760 g) was subjected to silica gel column chromatography (70–230 mesh, 3 kg, 15.0  100 cm) using a gradient of methylene chloride and methanol as the eluent to afford six fractions: f1 (106 g), f2 (5 g), f3 (27 g), f4 (300 g), f5 (152 g), and f6 (61 g). Fraction f1 (106 g) was allowed to stand at room temperature for several days and produced caffeine [24.7 g, white crystal, 1H NMR (300 MHz, DMSO-d6) d: 7.97 (1H, s), 3.86 (3H,s), 3.38 (3H,s), 3.19 (3H,s) 13C NMR (125 MHz, DMSO-d6) d: 154.5, 151.0, 148.1, 142.8, 106.6, 33.1, 29.3, 27.4] (Talebpour, Maesum, Jalali-Heravi, & Shamsipur, 2003). Fraction f2 was applied to a Sephadex LH-20 column (Ø = 2.0  80 cm) eluted with 50% methanol to give three subfractions. The third subfraction was rechromatographed on an RP-18 column to give EC [1.4 g, mp 241–243 °C, [a]D = 41.3 (C: 1.0, in MeOH), 1H NMR (300 MHz, DMSO-d6) d: 9.11 (1H, 5-OH), 8.90 (1H, s, OH-7), 8.81 (1H, s, OH-40 ), 8.72 (1H, s, OH-30 ), 6.89 (1H, br s, H-20 ), 6.66 (2H, br s, H-50 ,60 ), 5.89 (1H, d, J = 2.2 Hz, H-8), 5.72 (2H, d, J = 2.2 Hz, H-6), 4.74 (1H, br s, H-2), 4.66 (1H, d, OH-3), 4.01 (1H, t, J = 3 Hz, H-3), 2.57 (2H, m, H-4) 13 C NMR (125 MHz, DMSO-d6) d: 156.6 (C-5), 156.3 (C-7), 155.8 (C-9), 144.5(2) (C-30 ,40 ), 130.7 (C-10 ), 118.1 (C-60 ), 114.9 (C-50 ), 114.8 (C-20 ), 98.6 (C-10), 95.1 (C-6), 94.2 (C-8), 78.1 (C-2), 65.0 (C-3), 28.3 (C-4)] (Kwon, Lee, & Lee, 2002; Lee & Lee, 1995). Fraction f4 (34 g) was applied to a silica gel column (70–230 mesh, 980 g, 5.0  100 cm) and chromatographed using a gradient of methylene chloride and methanol to give six subfractions. The third subfraction was rechromatographed on an MCI gel column (Ø = 1.5  60 cm) using a gradient elution with methanol and water to afford EGC [1.65 g, mp 217–219 °C, [a]D = 60.0 (C: 1.0, in MeOH), 1H NMR (300 MHz, DMSO-d6) d: 9.11 (1H, 5-OH), 8.90 (1H, s, OH-7), 8.71 (2H, s, OH-30 ,50 ), 7.95 (1H, s, OH-40 ), 6.37 (2H, s, H-20 ,60 ), 5.88 (1H, d, J = 1.8 Hz, H-8), 5.71 (1H, d, J = 1.8 Hz, H6), 4.66 (1H, s, H-2), 4.62 (1H, d, OH-3), 4.12 (1H, m, H-3), 2.56 (2H, m, H-4) 13C NMR (125 MHz, DMSO-d6) d: 156.6 (C-9), 156.3 (C-7), 155.8 (C-5), 145.4(2) (C-30 ,50 ), 132.2 (C-40 ), 129.8 (C-10 ), 106.1(2) (C-20 ,60 ), 98.6 (C-10), 95.1 (C-6), 94.1 (C-8), 78.2 (C-2), 65.1 (C-3), 28.3 (C-4)], and EGCG [1.9 g, mp 211–213 °C, [a]D = 179 (C: 1.0, in MeOH), 1H NMR (300 MHz, DMSO-d6) d: 6.83 (2H, s, H-200 ,600 ), 6.42 (2H, s, H-20 ,60 ), 5.94 (1H, d, J = 2.0 Hz, H-8), 5.84 (1H, d, J = 2.0 Hz, H-6), 5.38 (1H, m, H-3), 4.97 (1H, s, H-2), 2.64–2.98 (2H, m, H-4) 13C NMR (125 MHz, DMSO-d6) d:

165.4 (C-700 ), 156.6(2), 155.8, 145.8 (2), 145.5(2), 138.7, 132.5, 128.8, 119.4, 108.8, 105.6, 97.5 (C-10), 95.6 (C-6), 94.5 (C8), 76.6 (C-2), 68.2 (C-3), 25.9 (C-4)] (Cho, An, & Choi, 1993). Fraction f5 (10 g) was applied to an RP-18 column and chromatographed using a gradient of methylene chloride and methanol to give five subfractions. The second (2.7 g) and fourth subfractions (400 mg) were combined and rechromatographed on a silica gel column using a gradient elution with methanol and water to afford ECG [970 mg, mp 253–255 °C [a]D = 160.6 (C: 1.0, in MeOH) 1H NMR (300 MHz, DMSO-d6) d: 6.88 (1H, dd, J = 7.2,1.8 Hz, H-20 ), 6.83 (2H, s, H-200 , 600 ), 6.76 (1H, dd, J = 7.2, 1.8 Hz, H-60 ), 6.76 (1H, d, J = 7.2 Hz, H-50 ), 5.95 (1H, d, J = 2.1 Hz, H-8), 5.84 (1H, d, J = 2.1 Hz, H-6), 5.36 (1H, m, H-3), 5.03 (1H, s, H-2), 2.65–2.98 (2H, m, H-4) 13C NMR (125 MHz, DMSO-d6) d: 165.3 (C-700 ), 156.6(2) (C-5,7), 155.7 (C-9), 145.5(2) (C-300 ,500 ), 144.8(2) (C30 ,40 ), 138.7 (C-400 ), 129.5 (C-10 ), 119.3 (C-100 ), 117.7 (C-60 ), 115.2 (C-50 ), 114.3 (C-20 ), 108.7(2) (C-200 ,600 ), 97.3 (C-10), 95.6 (C-6), 94.4 (C-8), 76.6 (C-2), 68.2 (C-3), 25.8 (C-4)] (Cho et al., 1993). 2.3. TOSC assay A slight modification of the method developed by Regoli and Winston (1999) was used to determine the TOSC of the catechins. Peroxyl radicals were generated by thermal homolysis of 60 mM ABAP at 35 °C in 100 mM potassium phosphate buffer, pH 7.4. Hydroxyl radicals were generated at 35 °C by the iron plus ascorbatedriven Fenton reaction. Final concentrations of ferric iron, ethylenediaminetetraacetic acid, and ascorbate were 9 lM, 18 lM, and 900 lM, respectively. Peroxynitrite was generated from the decomposition of 210 lM SIN-1 in the presence of 100 mM potassium phosphate buffer, pH 7.4, and 0.3 mM DTPA, at 35 °C. Reactions with 0.3 mM KMBA were carried out in 10-ml rubber septa-sealed vials in a final reaction volume of 1 ml. Ethylene production was measured by gas chromatographic analysis of 200 ll aliquots taken from the headspace of vials at indicated intervals during the course of the reaction. Total ethylene formation was quantified from the area under the kinetic curve. Samples were monitored in sequence at 12 min intervals over a time course of 60 min. Analyses were performed with a Shimadzu-2010 (Shimadzu Corp., Tokyo, Japan) gas chromatograph equipped with a DB05 capillary column (30 m  0.32 mm  0.25 lm) and a flame ionising detector (FID). The oven, injection, and FID temperatures were 60 °C, 180 °C, and 180 °C, respectively. Helium, at a flow rate of 30 ml/min, was used as the carrier gas. 2.4. Quantification of TOSC The TOSC value for each concentration of sample was calculated as follows:

R  SA TOSC ¼ 100  R  100 CA R R Here, SA and CA are the integrated areas from the sample reaction and control reaction, respectively. Thus, a sample with no oxy-radical scavenging capacity would give an area equal to R R the control ( SA/ CA=1) and a resulting TOSC value of 0. However, R as the SA approaches 0, the hypothetical TOSC value approaches 100. A specific TOSC (sTOSC) and relative TOSC (rTOSC) values were calculated using the method of Tung et al. (2008). Briefly, sTOSC values were obtained from the slope of the linear regression lines for the TOSC curves. rTOSC values were quantified by dividing the sTOSC value of the sample by that obtained of Trolox, as shown below:

rTOSC ¼

sTOSCðsampleÞ sTOSCðTroloxÞ

K.W. Kang et al. / Food Chemistry 121 (2010) 1089–1094

2.5. Statistical analysis The results are expressed as the means ± SD. Linear regression was calculated using GraphPad Prism, version 4.0. Specific TOSC values were analysed by one-way analysis of variance (ANOVA) using the Newman–Keuls multiple range test. The acceptable level of significance was established at P < 0.05 except when otherwise indicated. 3. Results and discussion Freshly collected green tea leaves were dried, macerated, and extracted with methanol. The extract was then partitioned in various solvents and the chemical constituents were separated by normal or reversed-phase flash chromatography. Based on the results of thin layer chromatography and NMR analysis, EC, EGC, ECG, and EGCG were isolated from the n-butanol extracts. Their structures were characterised by comparison of their physical properties, NMR spectra (1H and 13C NMR), and electrospray ionisation mass spectra with those in the literature (Fig. 1). E, EC, and ECG are compounds with a catechol structure in the B ring and EGC and EGCG are compounds with a pyrogallol structure. In our previous study, quantitative analysis of C, EC, EGC, ECG, and EGCG in green tea was carried out by high-performance liquid chromatography with gradient elution on ODS columns (Zhu et al., 2009). The contents of C, EC, EGC, ECG, and EGCG were 0.02 ± 0.01%, 0.79 ± 0.27%, 2.20 ± 0.77%, 1.22 ± 0.46%, and 5.03 ± 1.38%, respectively, of the dried green tea leaves (w/w). These results indicated that EGCG is a major component of green tea and suggested that pyrogalloltype catechins (EGC, EGCG) are more abundant than catechol-type catechins (C, EC, ECG) in green tea. Oxidative stress can be defined as an imbalance between oxidants or prooxidants and the antioxidant system leading to oxidative damage at critical sites in tissues and cells (Dröge, 2002). Under normal conditions, the levels of oxidants and antioxidants in humans are maintained in balance, which is important for sustaining optimal physiology. Oxidative stress has been implicated in a wide variety of pathological processes, including cancer, diabetes mellitus, steatohepatitis, atherosclerosis, neurological degeneration, and autoimmune disorders. Recently, growing interest has been expressed in the antioxidant activities of dietary components

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with the expectation that they may supplement the body’s defenses against various oxidant challenges (Panza et al., 2008). Many studies have indicated the antioxidant capacity of green tea (Fukushima et al., 2009), which appears to account for its beneficial effects against certain chronic human diseases related to oxidative stress shown in epidemiological studies (Kuriyama, 2008). In this study, the antioxidant capacities of catechins isolated from green tea against peroxyl radicals, hydroxyl radicals, and peroxynitrite were determined using the TOSC assay. The TOSC assay developed by Winston and collaborators is a method for evaluating antioxidant activity based on the inhibition of oxy-radical-induced production of ethylene gas from KMBA (Regoli & Winston, 1999). In our previous study, the antioxidant activities of a series of sugar alcohols were determined by the TOSC assay and cell-based assay (Kang, Kwak, Yun, & Kim, 2007). The specific TOSC values of erythritol, xylitol, sorbitol, and mannitol against peroxyl radicals, hydroxyl radicals, and peroxynitrite increased with increases in the number of aliphatic hydroxyl groups in the sugar alcohols. Cytotoxicity, glutathione depletion, and malondialdehyde elevation in cells treated with tert-butylhydroperoxide, a model oxidant for the induction of oxidative stress, were attenuated by 10 mM erythritol and completely prevented by 10 mM xylitol, 2 mM sorbitol, and 0.75 mM maltitol, a disaccharide polyol. These results indicated that the antioxidant activities of sugar alcohols are mediated by their aliphatic hydroxyl groups and that the TOSC assay is a reliable tool for quantitatively assessing the antioxidant potency of chemical substances. We have successfully evaluated the antioxidant capacities of plant extracts (Tai et al., 2009), isolated or synthetic compounds (Tung et al., 2008), and biological samples (Kim, Woodcroft, Oh, Abdelmegeed, & Novak, 2005; Kim et al., 2009) using the TOSC assay. In the present study, all samples including catechins and GA were analysed for at least five concentrations, and ethylene production was reduced in the presence of each sample in a concentration-dependent manner (data not shown). The slopes of the regression lines were calculated from the linear portion of TOSC vs. concentration of the catechins and GA used, and the sTOSC and rTOSC values against peroxyl radicals, hydroxyl radicals, and peroxynitrite are listed in Tables 1–3, respectively. Ethylene generation from KMBA oxidation upon thermal homolysis of ABAP was markedly inhibited by the catechins and

Fig. 1. Structures of the isolated catechins and gallic acid.

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Table 1 TOSC values of catechins and GA against peroxyl radicals. Compounds

Concentration (lM)

Table 2 TOSC values of catechins and GA against hydroxyl radicals.

TOSC value

sTOSC value (TOSC/lM)

rTOSC value

Compounds

Concentration (lM)

TOSC value

sTOSC value (TOSC/lM)

rTOSC value

C

0.25 0.5 1.0 2.5 5.0

8.8 18.1 35.8 53.8 72.5

35.9 ± 0.4a (R2 = 0.999)

8.5

C

12.5 25 50 100 250

3.6 8.2 16.5 32.9 46.2

0.331 ± 0.006a (R2 = 0.999)

3.1

EC

0.25 0.5 1.0 2.5 5.0

8.8 18.4 36.6 58.7 74.3

36.7 ± 0.7a (R2 = 0.999)

8.7

EC

12.5 25 50 100 250

3.7 8.4 17.9 34.4 47.5

0.348 ± 0.012a (R2 = 0.999)

3.3

EGC

0.25 0.5 1.0 2.5 5.0

1.2 3.5 10.2 26.3 52.7

10.8 ± 0.5b (R2 = 0.999)

2.6

EGC

12.5 25 50 100 250

2.1 3.7 7.5 14.5 36.4

0.145 ± 0.002b (R2 = 0.999)

1.4

ECG

0.25 0.5 1.0 2.5 5.0

8.2 21.9 41.6 61.5 70.2

42.5 ± 4.9c (R2 = 0.995)

10.1

ECG

12.5 25 50 100 250

3.7 7.6 18.2 36.8 47.6

0.374 ± 0.020a (R2 = 0.998)

3.5

EGCG

0.25 0.5 1.0 2.5 5.0

7.1 11.8 24.4 41.1 68.2

24.0 ± 2.2d (R2 = 0.997)

5.7

EGCG

12.5 25 50 100 250

2.5 4.4 10.9 22.2 47.8

0.225 ± 0.014c (R2 = 0.997)

2.1

4.7 13.3 21.3 40.0 56.7

7.7 ± 1.5e (R2 = 0.981)

1.8

GA

25 50 100 250 500

0.8 –0.7 10.4 27.3 50.2

0.106 ± 0.016d (R2 = 0.985)

1.0

GA

0.5 1.0 2.5 5.0 10.0

The TOSC values of each concentration are mean from two or three independent measurements. The sTOSC values were obtained from the slope of the linear regression lines for the TOSC curves, and the rTOSC values were determined by dividing the sTOSC value of the sample by that of Trolox. The specific TOSC values of Trolox against peroxyl radicals were 4.2 ± 0.3 TOSC/lM. Values with different letters are significantly different from each other (ANOVA followed by Newman–Keuls multiple range test, P < 0.05).

The TOSC values of each concentration are mean from two or three independent measurements. The sTOSC values were obtained from the slope of the linear regression lines for the TOSC curves, and the rTOSC values were determined by dividing the sTOSC value of the sample by that of Trolox. The specific TOSC values of Trolox against hydroxyl radicals were 0.107 ± 0.031 TOSC/lM. Values with different letters are significantly different from each other (ANOVA followed by Newman– Keuls multiple range test, P < 0.05).

GA (data not shown). ECG showed the greatest sTOSC value against peroxyl radicals followed in decreasing order by EC, C, EGCG, EGC, and GA (Table 1). All of the catechins and GA showed significantly greater antioxidant capacities against peroxyl radicals than Trolox, a positive antioxidant. All of the compounds examined, including catechins, GA, and Trolox, showed weak hydroxyl radical scavenging capacity compared to their peroxyl radical scavenging capacity (Table 2). The catechins and GA significantly inhibited ethylene gas production from hydroxyl radical-mediated KMBA oxidation (data not shown). C, EC, and ECG showed high antioxidant activity with specific TOSC values of 0.331 ± 0.006, 0.348 ± 0.012, and 0.374 ± 0.020 TOSC/lM, respectively, which was followed by EGCG, EGC, and GA (Table 2). The catechins examined in this study showed much higher oxyradical scavenging capacity than Trolox. The TOSC value of GA against hydroxyl radicals was comparable to that of Trolox. In the present study, the TOSC values of the catechins and GA against peroxynitrite were higher than those against hydroxyl radicals but less than those against peroxyl radicals (Table 3). Among the compounds examined, ECG and EGCG were shown to have the greatest antioxidant capacities, followed by EC, C, GA, and EGC (Table 3). All of the catechins and GA showed significantly greater antioxidant capacity against peroxynitrite than Trolox. Studies of the catechin structure–activity relationship with regard to antioxidant activity using DPPH radicals indicated that the antiradical ability according to the EC50 values was in the rank order of EGCG > ECG > EGC > EC (Sun & Ho, 2001). However,

antiradical efficiency according to EC50 and the time to reach the steady state was ECG > EGCG > EC > EGC (Sun & Ho, 2001). In contrast, EGC showed stronger scavenging capacity against superoxide anions generated by the hypoxanthine–xanthine oxidase system than that of ECG, but hydroxyl radical scavenging capacity was EGCG = ECG > EC > EGC (Nanjo, Mori, Goto, & Hara, 1999). These results suggest that the attachment of a galloyl moiety contributes to the strong radical scavenging activity of catechins, and that the oxy-radical scavenging capacity of catechins is dependent on the characteristics of oxy-radicals. In the present study, the TOSC values of EC against peroxyl radicals, hydroxyl radicals, and peroxynitrite were comparable to those of C, an EC epimer, indicating that the scavenging capacity of the catechins is not dependent on their structure. These results were consistent with those of previous studies using DPPH radicals (Nanjo et al., 1996) and superoxide anions (Nanjo et al., 1999). The scavenging capacities of galloylated catechins, such as ECG or EGCG, were stronger than those of non-galloylated catechins, such as EC or EGC. These results indicated that the presence of the gallate group at the three position plays a critical role in oxy-radical scavenging capacity. Several studies have indicated the importance of the gallate group in the antioxidant activities of catechins (Guo et al., 1999; Sun & Ho, 2001). Moreover, the present study showed that GA is a more potent scavenger of peroxyl radicals and peroxynitrite than Trolox as a positive control, and that GA showed hydroxyl radical scavenging activity comparable to that of Trolox.

K.W. Kang et al. / Food Chemistry 121 (2010) 1089–1094 Table 3 TOSC values of catechins and GA against peroxynitrite. Compounds

Concentration (lM)

TOSC value

sTOSC value (TOSC/lM)

rTOSC value

C

0.25 0.5 1.0 2.5 5.0

5.2 11.4 20.1 34.0 41.3

20.2 ± 2.2a (R2 = 0.994)

7.4

EC

0.25 0.5 1.0 2.5 5.0

5.2 11.2 20.4 35.6 44.4

20.5 ± 1.6a (R2 = 0.997)

7.5

EGC

0.25 0.5 1.0 2.5 5.0

–0.2 0.7 4.4 12.3 20.1

4.3 ± 0.7b (R2 = 0.980)

1.6

ECG

0.25 0.5 1.0 2.5 5.0

6.1 11.6 23.7 47.0 52.5

23.6 ± 0.6c (R2 = 0.999)

8.6

EGCG

0.25 0.5 1.0 2.5 5.0

6.2 11.6 22.6 43.1 53.3

22.5 ± 0.8c (R2 = 0.999)

8.2

0.5 5.6 12.6 24.1 57.4

5.8 ± 0.7d (R2 = 0.992)

GA

0.5 1.0 2.5 5.0 10.0

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anthocyanin-like compound produced from EC by oxy-radical oxidation functioned as an antioxidant and that active oxygen may be produced from EGC. Roginsky (2003) suggested that the elevated antioxidant capacity of EC compared with EGC can be explained by the contribution of active compounds produced from EC, most likely by dimers. These results suggest that the antioxidant activities of catechins may be determined by the reactivity of their active products as well as original catechins. In the present study, ECG showed the highest sTOSC against peroxyl radicals, hydroxyl radicals, and peroxynitrite, while EGC was the least effective among the five catechins examined. However, multiplication of the sTOSC value by catechin content in green tea suggests that EGCG may be a major contributor to the oxy-radical scavenging activity of green tea, followed by ECG, EC, EGC, and C. Note also that the antioxidant capacities measured in vitro are not necessarily consistent with their effects in vivo. Characterisation of antioxidant components in green tea and data regarding their pharmacokinetic properties, including bioavailability, tissue distribution, metabolism, and excretion, and their effects on antioxidant enzyme expression via transcriptional and/or posttranscriptional regulation are needed to clarify the protective role of green tea in vivo.

Acknowledgements 2.1

The TOSC values of each concentration are mean from two or three independent measurements. The sTOSC values were obtained from the slope of the linear regression lines for the TOSC curves, and the rTOSC values were determined by dividing the sTOSC value of the sample by that of Trolox. The specific TOSC values of Trolox against peroxynitrite were 2.74 ± 0.32 TOSC/lM. Values with different letters are significantly different from each other (ANOVA followed by Newman–Keuls multiple range test, P < 0.05).

The sTOSC values showed that EC and ECG, which have an ortho-dihydroxyl group (catechol) in the B ring, had more potent scavenging capacity against peroxyl radicals and hydroxyl radicals than EGC and EGCG, respectively, which have an ortho-trihydroxyl group (pyrogallol) in the B ring. In addition, EC showed more potent peroxynitrite scavenging capacity than EGC, although no significant differences were observed in the peroxynitrite scavenging capacity between ECG and EGCG. These results showed that insertion of an additional hydroxyl group at the 50 position in the B ring attenuates the oxy-radical scavenging capacity of catechins. The presence of at least two hydroxyl groups in the B ring has been suggested to be important for the antioxidant activities of catechins (Guo et al., 1999; Sun & Ho, 2001). Nanjo et al. (1996, 1999) reported that the ortho-trihydroxyl group in pyrogallol-type catechin increased the scavenging efficiency against DPPH radicals and superoxide anions, but that EC and C were more potent scavengers of hydroxyl radicals generated by the Fenton reaction than EGC. Based on the O–H bond dissociation enthalpies, the reactivity of the hydroxyl group in pyrogallol was reported to be higher than that in catechol, suggesting that the ability to donate a hydrogen atom is increased by the presence of ortho- and para-hydroxyl groups (Thavasi, Leong, & Bettens, 2006). Kondo, Kurihara, Miyata, Suzuki, and Toyoda (1999) determined the antioxidant activities of catechins during lipid peroxidation induced by ABAP. In their study, EC had a longer inhibition period, but EGC was the least effective among the catechins and rapidly increased peroxides after an initial inhibition period. The authors suggested that an

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MEST) (Grant R11-2002-100-00000-0).

References Amit, T., Avramovich-Tirosh, Y., Youdim, M. B., & Mandel, S. (2008). Targeting multiple Alzheimer’s disease etiologies with multimodal neuroprotective and neurorestorative iron chelators. Faseb Journal, 22(5), 1296–1305. Babu, P. V., & Liu, D. (2008). Green tea catechins and cardiovascular health: An update. Current Medicinal Chemistry, 15(18), 1840–1850. Cho, Y. J., An, B. J., & Choi, C. (1993). Isolation and enzyme inhibition of tannins from Korean green tea. Korean Biochemical Journal, 26, 216–223. Dröge, W. (2002). Free radicals in the physiological control of cell function. Physiological Reviews, 82(1), 47–95. Fukushima, Y., Ohie, T., Yonekawa, Y., Yonemoto, K., Aizawa, H., Mori, Y., et al. (2009). Coffee and green tea as a large source of antioxidant polyphenols in the Japanese population. Journal of Agricultural and Food Chemistry, 57(4), 1253–1259. Guo, Q., Zhao, B., Shen, S., Hou, J., Hu, J., & Xin, W. (1999). ESR study on the structure–antioxidant activity relationship of tea catechins and their epimers. Biochimica et Biophysica Acta, 1427(1), 13–23. Kang, K. W., Kwak, S. H., Yun, S. Y., & Kim, S. K. (2007). Evaluation of antioxidant activity of sugar alcohols using TOSC (total oxy-radical scavenging capacity) assay. Journal of Toxicology and Public Health, 23(2), 143–150. Kim, S. K., Seo, J. M., Chae, Y. R., Jung, Y. S., Park, J. H., & Kim, Y. C. (2009). Alleviation of dimethylnitrosamine-induced liver injury and fibrosis by betaine supplementation in rats. Chemico-Biological Interactions, 177(3), 204–211. Kim, S. K., Woodcroft, K. J., Oh, S. J., Abdelmegeed, M. A., & Novak, R. F. (2005). Role of mechanical and redox stress in activation of mitogen-activated protein kinases in primary cultured rat hepatocytes. Biochemical Pharmacology, 70(12), 1785–1795. Klaunig, J. E., Xu, Y., Han, C., Kamendulis, L. M., Chen, J., Heiser, C., et al. (1999). The effect of tea consumption on oxidative stress in smokers and nonsmokers. Proceedings of the Society for Experimental Biology and Medicine, 220(4), 249–254. Kondo, K., Kurihara, M., Miyata, N., Suzuki, T., & Toyoda, M. (1999). Mechanistic studies of catechins as antioxidants against radical oxidation. Archives of Biochemistry and Biophysics, 362(1), 79–86. Kuriyama, S. (2008). The relation between green tea consumption and cardiovascular disease as evidenced by epidemiological studies. The Journal of Nutrition, 138(8), 1548S–1553S. Kwon, Y. M., Lee, J. H., & Lee, M. W. (2002). Phenolic compounds from barks of Ulmus macrocarpa and its antioxidative activities. Korean Journal of Pharmacognosy, 33, 404–410. Lee, Y. A., & Lee, M. W. (1995). Tannins from Rubus coreanum. Korean Journal of Pharmacognosy, 26, 27–30. Leenen, R., Roodenburg, A. J., Tijburg, L. B., & Wiseman, S. A. (2000). A single dose of tea with or without milk increases plasma antioxidant activity in humans. European Journal of Clinical Nutrition, 54(1), 87–92.

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K.W. Kang et al. / Food Chemistry 121 (2010) 1089–1094

Mukhtar, H., & Ahmad, N. (1999). Mechanism of cancer chemopreventive activity of green tea. Proceedings of the Society for Experimental Biology and Medicine, 220(4), 234–238. Na, H. K., & Surh, Y. J. (2008). Modulation of Nrf2-mediated antioxidant and detoxifying enzyme induction by the green tea polyphenol EGCG. Food and Chemical Toxicology, 46(4), 1271–1278. Nanjo, F., Goto, K., Seto, R., Suzuki, M., Sakai, M., & Hara, Y. (1996). Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical. Free Radical Biology and Medicine, 21(6), 895–902. Nanjo, F., Mori, M., Goto, K., & Hara, Y. (1999). Radical scavenging activity of tea catechins and their related compounds. Bioscience, Biotechnology and Biochemistry, 63(9), 1621–1623. Panza, V. S., Wazlawik, E., Ricardo Schütz, G., Comin, L., Hecht, K. C., & da Silva, E. L. (2008). Consumption of green tea favorably affects oxidative stress markers in weight-trained men. Nutrition, 24(5), 433–442. Regoli, F., & Winston, G. W. (1999). Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite, peroxyl radicals, and hydroxyl radicals. Toxicology and Applied Pharmacology, 156(2), 96–105. Roginsky, V. (2003). Chain-breaking antioxidant activity of natural polyphenols as determined during the chain oxidation of methyl linoleate in Triton X-100 micelles. Archives of Biochemistry and Biophysics, 414(2), 261–270. Sun, T., & Ho, C. T. (2001). Antiradical efficiency of tea components. Journal of Food Lipids, 8, 231–238.

Tai, B. H., Jung, B. Y., Cuong, N. M., Linh, P. T., Tung, N. H., Nhiem, N. X., et al. (2009). Total peroxynitrite scavenging capacity of phenylethanoid and flavonoid glycosides from the flowers of Buddleja officinalis. Biological & Pharmaceutical Bulletin, 32(12), 1952–1956. Talebpour, Z., Maesum, S., Jalali-Heravi, M., & Shamsipur, M. (2003). Simultaneous determination of theophylline and caffeine by proton magnetic resonance spectroscopy using partial least squares regression techniques. Analytical Sciences, 19(7), 1079–1082. Tedeschi, E., Suzuki, H., & Menegazzi, M. (2002). Antiinflammatory action of EGCG, the main component of green tea, through STAT-1 inhibition. Annals of the New York Academy of Sciences, 973, 435–437. Thavasi, V., Leong, L. P., & Bettens, R. P. (2006). Investigation of the influence of hydroxy groups on the radical scavenging ability of polyphenols. The Journal of Physical Chemistry A, 110(14), 4918–4923. Tung, N. H., Ding, Y., Kim, S. K., Bae, K., & Kim, Y. H. (2008). Total peroxyl radicalscavenging capacity of the chemical components from the stems of Acer tegmentosum Maxim. Journal of Agricultural and Food Chemistry, 56(22), 10510–10514. Zhu, H., Kim, J. S., Park, K. L., Cho, J. W., Kim, Y. S., Kim, J. W., et al. (2009). Estimation of harvest period and cultivated region of commercial green tea by pattern recognition. Yakhak Hoeji, 53(2), 51–59.